Maraia R J, Sasaki-Tozawa N, Driscoll C T, Green E D, Darlington G J
Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
Nucleic Acids Res. 1994 Aug 11;22(15):3045-52. doi: 10.1093/nar/22.15.3045.
Ro ribonucleoproteins (RNP) constitute a class of evolutionarily conserved small cytoplasmic (sc) RNPs whose functions are unknown. In human cells four distinctive scRNAs designated hY1, hY3, hY4 and hY5 are synthesized by RNA polymerase III (pol III) and accumulate as components of Ro scRNPs. The previously isolated hY1 and hY3 genes contain upstream sequences similar to the class III promoters for U6 and 7SK snRNAs. Additional mammalian Y scRNA genes have been refractory to cloning due to interference from numerous hY-homologous pseudogenes and studies of hY RNA genes have been sparse. Although homologs of hY1 and hY3 RNAs exist in rodent cells, the smaller Y4 and Y5 RNAs do not which has allowed us to localize the hY4 scRNA gene to human chromosome 7 by assaying for its transcript in rodent X human somatic cell hybrids (SCH). A chromosome 7-enriched yeast artificial chromosome (YAC) library was then screened and the authentic hY4 sequence was isolated by strepavidin--biotin-mediated hybrid-selection followed by poly(dA)-tailing and hemispecific PCR. The region upstream of the hY4 sequence contains a TATAAAA motif centered at -26, a candidate proximal sequence element at -63, and three octamer-like sequences located between -260 and -200. hY4 RNA is readily detectable on Northern blots after transient transfection of the hY4 gene into mouse cells but not after transfection of a construct in which the 5' flanking region was deleted. SCHs and chromosome 7-enriched YACs were used to demonstrate that all four hY RNA genes reside on human chromosome 7.
Ro核糖核蛋白（RNP）构成了一类进化上保守的小细胞质（sc）RNP，其功能尚不清楚。在人类细胞中，四种独特的scRNA，即hY1、hY3、hY4和hY5，由RNA聚合酶III（pol III）合成，并作为Ro scRNP的组分积累。先前分离的hY1和hY3基因含有与U6和7SK snRNA的III类启动子相似的上游序列。由于众多hY同源假基因的干扰，其他哺乳动物Y scRNA基因难以克隆，并且对hY RNA基因的研究也很少。尽管hY1和hY3 RNA的同源物存在于啮齿动物细胞中，但较小的Y4和Y5 RNA不存在，这使我们能够通过在啮齿动物X人类体细胞杂种（SCH）中检测其转录本来将hY4 scRNA基因定位到人类染色体7上。然后筛选了一个富含染色体7的酵母人工染色体（YAC）文库，并通过链霉亲和素 - 生物素介导的杂交选择，随后进行聚（dA）加尾和半特异性PCR分离出真实的hY4序列。hY4序列上游的区域包含一个以-26为中心的TATAAAA基序、一个位于-63的候选近端序列元件，以及位于-260和-200之间的三个八聚体样序列。将hY4基因瞬时转染到小鼠细胞后，在Northern印迹上很容易检测到hY4 RNA，但转染缺失5'侧翼区域的构建体后则检测不到。使用SCH和富含染色体7的YAC来证明所有四个hY RNA基因都位于人类染色体7上。
